With regard to cancer therapy, in recent years, much attention has been paid to gene therapy of cancer wherein a foreign gene is introduced into the cell to treat cancer. Gene therapy of cancer includes: the gene therapy that targets oncogenes and/or tumor suppressor genes wherein the action of oncogenes is suppressed or the denatured tumor suppressor genes are reactivated; the suicide gene therapy wherein a gene for drug metabolizing enzyme that is not originally present in human cells is introduced into tumor cells and then a prodrug for cancer therapy that is activated by the said enzyme is administered so as to kill only the cells into which the gene for a drug metabolizing enzyme has been introduced; the immunological gene therapy wherein cytokine genes are etc. introduced into the cell so as to enhance the immunological functions inherent to human and thereby to treat cancer; a gene therapy wherein a tumor-specific promoter is inserted upstream of adenovirus E1A or E1B gene that is one of the early genes and is essential for the propagation of adenoviruses so as to construct an oncolytic virus that specifically propagates in the integrated tumor cells, and tumor cells are specifically killed by this virus, and the like.
In such gene therapies, specific expression of the desired gene in tumor cells or tumor tissues has an important meaning from the viewpoint of efficiency and safety of gene therapy, and is one of the challenges encountered by gene therapy today. In order to permit such tumor-specific gene expression, it is important to develop a promoter that regulates the expression of the introduced gene in a tumor-specific manner.
As tumor-specific promoters, there are known α-fetoprotein promoter, carcinoembryonic antigen (CEA) promoter, prostate specific antigen promoter and the like. However, these promoters lack flexibility since the scope in which they can be applied is limited and the promoter activity is not high. Thus, the gene therapy using these promoters has a very limited scope of application.
On the other hand, a new retinoic acid-responsive growth/differentiation factor, midkine, a heparin-binding protein, was discovered from teratoma cells, and the gene thereof has been cloned (Kodomatsu, K. et al., Biochem. Biophys. Res. Commun. 151:1312–1318, 1988). Though the biological functions of the protein have not been fully elucidated, its expression frequency and expression level are high in many kinds of human gastrointestinal cancers (colon cancer, pancreatic cancer, hepatic cancer etc.), lung cancer, breast cancer, neuroblastoma, brain tumor etc. (Tsutui, J. et al., Cancer Res., 53:1281–1285, 1993; Aridome, K. et al., Jpn. J. Cancer Res., 86:655–661, 1995; Muramatsu, H. et al., J. Biochem., 119:1171–1175, 1996; O'Brien, T. et al., Cancer Res., 56:2515–2518, 1996).
c-erbB-2 (HER2/neu) belongs to the EGF receptor family and has a tyrosine kinase activity. Although this gene is expressed slightly in the normal epithelial cells, it is highly expressed in breast cancer and other cancers such as esophageal cancer, gastric cancer and ovarian cancer (Slamon, D J et al., Science, 244:707–712, 1989; Hynes, N E et al., Biochim. Biophys. Acta, 1198:165–184, 1994). Furthermore, it has been reported that its high expression in breast cancer is associated with resistance to anti-cancer agents and is thereby a factor responsible for poor prognosis (Hynes, N E et al., Biochim. Biophys. Acta, 1198:165–184, 1994).
The promoter regions of the midkine gene and the c-erbB-2 gene are also being extensively studied.
Thus, it has been demonstrated that 2.3 kb in the 5′-end upstream region of the midkine gene can be used as a promoter that induces the transcription activity of the gene in the tumor cells and that it can be used as a promoter for the suicide gene therapy in which the herpes simplex virus-thymidine kinase (HSV-TK), a drug-metabolizing enzyme gene, and gancyclovir, a prodrug for cancer therapy, were combined (Miyauchi, M. et al., Jpn. J. Cancer Res., 90:469–475, 1999; Adachi, Y. et al., Mol. Ther., 1: S238, 2000). However, the tumor-specificity and the promoter activity of the 2.3 kb at the 5′-end upstream region of the midkine gene is not fully high.
Although the promoter region of the c-erbB-2 gene has also been analyzed, previous studies used animal species other than humans and tumor cells other than breast cancer and thus the identification of the minimal promoter region differed with different reports (Ishii, S. et al., Proc. Natl. Acad. Sci. USA, 84:4374–4378, 1987; Hudson, L G et al., J. Biol. Chem., 265:4389–4393, 1990; Hollywood, D P et al., EMBO J., 12:2369–2375, 1993; Scott, G K et al., J. Biol. Chem., 269:19848–19858, 1994; Benz, C C et al., Oncogene, 15:1513–1525, 1997; Grooteclaes, M. et al., Cancer Res., 59:2527–2531, 1999). However, there are many reports which described that, when the base at position 1259 of the c-erbB-2 promoter gene (Accession No. J05264) registered at the NCBI GeneBank is set as the transcription initiation point +1, a promoter activity was mapped in the −700 bp (−662/+38) region or the −533 bp (−435/+38) region, and it has been recognized that the activity lies approximately in this range. There are also reports which described that a promoter activity lied in −213/+38 or −87/+38 region, but their tumor specificity has not been studied and that the general promoter region of the c-erbB-2 gene but not tumor-specific regions has merely been examined using tumor cells. In fact, all of the previous reports that used the c-erbB-2 promoter for the expression of suicide genes have used the 533 bp (−465/+38) or longer regions containing the 533 bp (Harris, J D et al., Gene Ther., 1:170–175, 1994; Ring, C J A et al., Gene Ther., 3:1094–1103, 1996; Takakuwa, K. et al., Jpn. J. Cancer Res., 88:166–175, 1997; Pandha, H S et al., J. Clin. Oncol., 17:2180–2189, 1999). Furthermore, there is also a report that the 257 bp (−256/+1) region of the c-erbB-2 gene has a promoter activity specific to cancer (Japan Society of Gene Therapy, Abstract, issued on Jul. 18, 1999, page 98; Japan Society of Gene Therapy, Abstract, issued on Jul. 28, 2000, page 65). However, this region does not have fully high tumor-specificity or high promoter activity, either.
Thus, there has been a need for the development of tumor-specific promoters that have fully high tumor-specificity and high promoter activity, and that can be effectively used in gene therapy.